NEK7/NLRP3炎症小体活化通路基因型-表型与冠心病MACEs关联的队列研究

The major adverse cardiac events (MACEs) are the direct and/or indirectly causes of the death in the coronary heart disease (CAD) patients, of which about 1/4 of the deaths could be avoided. The recent studies revealed that NEK7 kinases could activate the NLRP3 inflammasome and then initiated the downstream cytokines to release, which were the important reasons for the instability of plaque rupture and thrombosis. Our previous study have found that, the genetic variants in Interleukin-1 B (IL-1B) gene, which encode the IL-1 downstream cytokines in the inflammasome activation pathway, are associated with MACEs and the increasing transcriptional activity of IL-1B. However, due to the interference of various potential confounding factors, we could not infer the relationships between cytokine phenotype and MACEs. Therefore, in this study, we will combine the latest epidemiological method - Mendelian randomization method and the cohort follow-up study design, with genotypes as instrumental variables, by constructing the relationships between genotypes and phenotypes, genotypes and MACEs outcomes, to infer the relationships between the relative cytokine phenotypes(NEK7, NLRP3, Caspase-1 and IL-1/18) and MACEs outcomes. And also, we have drawn a directed acyclic graph (DAG) to obtain a minimal sufficient adjustment set of variables (MSAS), which is used to adjust the potential confounding factors. Besides, we will test the effects on the stability of plaque of the genetic variations, using the cytological experiments and ApoE(-/-) gene knockdown animal model. This study will further elucidate the molecular biology mechanism of atherosclerotic plaque instability, provide new molecular biomarkers for the prediction of MACEs in coronary heart disease patients and promote the practice of CAD precision medicine.

新近研究发现NEK7可激活NLRP3炎症小体，并促进下游细胞因子的释放，可能是促使斑块不稳定/破裂进而发生冠心病不良心血管事件(MACEs)的始动因子。我们前期研究亦发现，炎症小体活化通路下游基因IL-1B多态性和冠心病MACEs的发生有关，并且突变可以带来转录活性的增加。然而，因各种潜在混杂因素的干扰我们尚无法推断细胞因子表型与MACE的关联。因此本研究结合国际流行的孟德尔随机化法，采用队列随访设计，以基因型为工具变量，通过构建基因型-表型，基因型-MACEs的关联来推断NEK7/NLRP3炎症小体活化通路细胞因子表型与MACEs发生的关联，同时绘制有向非循环图（DAG）获得潜在混杂因素的调整模型，使用细胞学实验和模式动物验证遗传变异对斑块稳定性的影响。本研究可进一步阐明动脉粥样斑块不稳定的分子生物学机制，并为预测冠心病病例发生MACEs提供新的分子标志物，促进冠心病精准医学的实践。

冠心病是一种血管炎症性疾病，包括炎症小体在内的多种炎症因子参与冠状动脉斑块的进展、破裂及血栓形成、内皮功能障碍等生理病理过程。本研究采用病例对照和前瞻性随访研究设计，选择炎症小体(NLRP3 rs10754558C/G)及其下游炎症因子的基因变异(IL-18 rs187238(G/C)和 rs1946518(C/A))作为工具变量，结合血清学相关表型NLRP3、IL-18水平的采集，采用孟德尔随机化研究设计，验证基因型-表型、基因型-疾病结局之间的关联，发现NLRP3水平与冠心病的发生风险存在因果关联；采用中介作用分析方法探讨炎症因子（IL-6, hs-CRP）在血脂代谢紊乱(TG、HDLC水平异常)与冠心病关联中的中介作用，发现IL-6在血脂异常与冠心病发生中起部分中介作用（占比为9.43%）。在吸烟对冠心病的影响中，17.52%是由TG和HDL-C异常介导的，同时TG/HDL-C比值也存在部分中介效应(占比28%)；冠心病病例5年随访研究中发现炎症小体相关基因（IL1B, IL12B）变异与冠心病不良预后MACEs发生风险增加有关，报告基因功能学验证了突变位点带来的功能学效应。本研究是孟德尔随机化法和中介作用分析方法在冠心病因果关联研究中的崭新尝试，科学构建了炎症小体暴露标志物与冠心病发生的因果关联，为全面探讨炎症小体及其下游炎症因子在冠心病发生发展中的作用机制提供证据。